16alpha-hydroxy-steroids



United States Patent O 3,188,325 1 16u-HYDROXY-STEROIDS Alba Maria Amici, Via Morigi 13; Maria Luisa- Bianchi, Via S. Eufemia 2; Renato Modelii, Via E. Filiberto 4;

ice

: DESCRIPTION. AND IDENTIFICATION OF THE STRAIN The new microorganism Nocardia italica isolated from Celestine Spalla, Via Tagiura 16; Aurelio di Marco, 5 a soil sample has the following morphologic, cultural and Via Argenta and Marcello Gaetani, Largo Rio de bi h i l. characteristics. 1 V Jflfleiro all Of Milan, y On the usual culture media the microor f ganism orms No zgi l ig 5 fi fi' gi at first a spreadeout mycelium with very branched, vnonalms P110 fl ca on y septate hyphaen After 34 days the formation of trans- 13,227/ 61, Mar. 14, 1962, 5,088/ 62 7 5 (312mm (Cl. 260 397 4) 10 versal septa is observed and afterwards the mycelr-um fragments into portions having various shapes and lengths Our invention relates to, a new method for the prepara- (4-8 1). The hyphae thickness ranges from 0.5 to 0.7 tion of 16a-hydroxy-steroids and to new therapeutically On liquid media, the fragmentation occurs within 2 or 3. useful products obtained thereby. Our invention has as days; V and Y forms are frequent, while coccoid forms are an object a microbiological process to introduce a hyabsent; Noaerial mycelium is observed in any medium. droxy group into the l6zx-position of a steroid molecule, The mycelium is gram-positive and partially acid fast. using the new microorganism Nocardia italica n.sp. A In the following table the cultural. characteristics are further object is the providing of some new l6m-hydroxy-V reported; the observations have been carried out after 10- 17a-methyl-androstenols of the normal and l9-nor-series, 20 152025 days of incubation at 28 C. The cultures'have obtained by the process of the invention. been repeated six times for each type of medium.

Tablel Vegetative mycelium Culture medium Soluble pigments Remarks J I. Growth 'Color Czapek a ar su hr I Calm-lo Yeast agar Abundant wrinkled growth. Yellowish Malt agar do do Potato agar Abundant growth with Egg-yellowish";

relieved folds. Jensen agar Scattered small colonies Color-1e s Nutritive agar with glyo- Abundant, wrinkled,,pasty Egg-yellowish Slighty yel1ow'lsh er consistence. Bennet agar Abundant wrinkled growth, Orange-yellowish" pasty consistence. Glucose asparagine again--. Good, uniform growth. Egg-yellowish pasty consistence. Sabouraud agar Abundant, wrinkled Brown-yellowish (Light-brown) growth, pasty consistence. Starch agar Good, nif rm Or n It hydrolyzes abundantly. Yeast broth Fltaky colonies at the bot- Yellowish om. v Peptone broth with potas Flaky colonies at the bot- Nitrates not reduced.

sium nitrate. in end.

Abundantwith large iolds Egg-yellowish Slight ring-p Yellow Peptonization and coagulation in 15 days.

Scanttsmall colonies at the Slight fluidification.

nerea, Didimellzz voa'akii, and Streptomycfes mediocidicus, Streptomyces halstedz'i. P

The new microorganism employed in the process of the present invention, named Nocardia italica n.sp., which has been deposited with the National Collection of Industrial Bacteria receiving the index number NJC.I.B. 9386 and at the Institute of Microbiology of Rutgers University receiving the number 3856, belongs to the genus Nocardia Trevisan whose capacity of hydroxylating a steroid in the la-position has been hitherto unknown. The use of Nocardia itlacia gives high transformation yields (70 80%) and high steroid concentration (0.1%) in the culture broth.

- The biochemical characteristics of the microorganism N ocardia inalica are the following:

Gelatine: slow and slight fluidification;

Nitrates: no reduction to nitrites;

Milk: coagulation and peptonizationj Starch: abundant hydrolysis; and

Acids production: positive from maltose, d-xylose, dmannose, mannitol, glycerol, glucose, levulose;fnega- V the from lactose, adonitol, d-sorbitol, l-arab inose, sac

charose, trehalose, reflinose, esculine.

The descriptionof the tested microorganism correspondsto that of the genus Nocardia Trevisan, which has been referred to in Bergeys Manual of Determinative Bacteriology (seventh edition, 1957, pages 713-715), so that we may conclude that the microorganisms of the present invention belongs to the genus Nocardia Trevi-san.

The analytic key of the species of genus Nocardia Waksman and Henrici (Waksman and Lechevalier: Guide to the classification and identification of the Actinomyceteslaud their antibiotics, 1953) indicates for the microorganism a systematic position close to Nocardia flava .(Krassilnikev, 1938) Waksman and Henrici, 1948, from which it differs, however, because it is partially acid-resistant, because it liquefies gelatine and peptonizes milk and because the mycelium doesonot form coccoid fragments. It is also close to Nocardia lutea (Christopherson and Archibald, 1918), from which it differs, however, because it liquefies gelatine, does not show pink or red colored mycelium is any tested media and does not form aerial myceliurn on potato plugs. Therefore, we conclude that the tested microorganism has neither been isolated nor described before.

The stock cultures of N. italica may be stored by lyophylization, milk or milk serum being the suspending medium. It may also be kept by succesive transfers on glucose potato agar. Our invention provides a microbiological process of preparing 16a-hydroxy-steroids which comprises treating a steroid compound having a methylenic group in the 16=positi0n with a culture produced by fermenting the new microorganism Nocardia italica n.sp. or a mutant thereof in asterilized liquid nutritional medium containing assimilable sources of carbon and nitrogen at from 24 to 30 C. at a pH of from 6 to 7.5, and extracting the 16u-hydroxy-steroid produced.

The process of our invention may be applied to a great number of steroid compounds, more generally to all steroid compounds having from 19 to 21 carbon atoms which may contain in their molecules, for example, one or more double bonds in the 1-, 4-, 6- or 9 (11) -positions, halogen atoms in the 6- or 9-position, one or more hydroxy or alkoxy groups in the 9-, 11-, 17- or 2l-positions, one or more keto groups in the 3-, 11-, 17- or 20-positions, one or more alkyl groups in the 6- or 17-positions.

The new compounds of the invention are:

l6 a-hydroxy-steroids of the general formula:

om Yon substances usually employed. The inorganic salts may be sodium, potassium, magnesium, sulfates, phosphates, chlo rides and calcium carbonate. It is also useful to add surfactants, such as available under the trademarks Tween 80 and Span.

p The microorganism grows in submerged and aerated conditions, in shake flasks or in fermenters at a tempera ture ranging from 24 and 30, preferably at 28 C. The pH of the medium should range between 6 and 7.5, preferably at 6.8. During fermentation the pH is quite constant.

The transformation of the starting 16-desoxy-steroid into the corresponding 16a-hydroxy-derivative during fermentation is checked by chromatography on thin lay ers. The chromatography on thin layers is described in the literature by Egon Stahl (Pharmac. Weekblad, 92,. 1957, page 829; Chemiker Ztg., 82, 1958, page 323) and by N. J. D. Van Dam et al. (I. Chromatography 4, 1960, page 26). Chromatography on thin layers is also very suitable for a quantitative analysis by comparing the spot intensity with that of standard steroid solution of known concentrations. The steroid substance contained in the culture broth (both the reacted products and the unre acted starting material) is recovered by known extraction processes. Preferably the extraction is carried out in the following manner.

The fermentation broth is mixed with a siliceous earth material, such as Supercel or Celite (registered trademarks) and the resulting mixture is warmed up to 50- 60 C. for a few minutes, whereupon the whole is filtered, and the filtration cake is washed with water at 50 C. and discarded. The filtrate is extracted with an organic solvent, such asethyl acetate, chloroform or methylene dichloride, the extracts are combined and washed with a subjected to the fermentative process by using N. italica are: progesterone, cortisone, hydrocortisone, testosterone, Reichsteins Compound S, desoxycorticosterone, 9a-fil10- ro-hydrocortisone, Qa-fiUOIO-PIEdHiSOlOHe, 4-androstene- 3,17-dione, 9a-chloro-hydrocortisone, 9a-bromo-hydrocortisone, ll-epi-hydrocortisone, 9a-fiuoro-cortisone, 9amethoxy-cortisone, 6a-fluoro-hydrocortisone, 6a-methylhydrocortisone, 4,6 pregnadiene 11p,17a,21 triol-3,20- dione, 17oc-methyl-19-nor-testosterone, 4-hydroxy-17amethyl-testosterone, 4-hydroxy-17u-methyl-19-nor-testosterone.

The process of our invention introduces a hydroxy group into the .16a-position of steroids and consists in fermenting N. italica in suitable media and conditions, and submitting the starting 16-desoxy-steroid to the action of the microorganism or to the enzymes thereof. Said steroid may be added to the cultures either at the beginning, or during or a the end of growth, either in crystalline form or dissolved in suitable solvents such as methyl alcohol, ethyl, alcohol, acetone or dimethylformamide for example.

The culture media consist of a source of carbon, of nitrogen and of inorganic salts. The carbon source may be constituted by starch, dextrin, saccharose, glucose, mal tose, glycerin, vegetable oils, cereal or legume flour, corn steep liquor and/or other substances usually employed. The nitrogen source, besides the above-cited complex substances containing nitrogen, may be constituted by casein, ammonia salts, peptones, meatand fish-flour or other saturated solution of sodium bicarbonate, then with Water to neutrality. The solvent is evaporated and the transformaion product is recovered in the usual manner. The

transformation product often crystallizes during concentration. It may be convenient to recrystallize the residue from acetone-petroleum ether or from other organic solvents. In other cases, column chromatography over silica gel may be used to obtain the compound in pure form.

Instead of submitting the crude products of the present invention to the above-cited purifying process, they may be acylated in 4,16-position or in the 16-position with the anhydride or the chloride of an aliphatie, cycloaliphatic-, aromatic-acid having from 1 to 9 carbon atoms in the optional presence of tertiary amines such as pyridine or the analogues thereof, dimethylaniline, trimethylamine, and then they are isolated and purified. Typical examples of acyl derivatives, prepared according to the present invention, are the derivatives of the following acids: acetic, propionic, cyclopentylpropionic, benzoic, phenylpropionic.

We have also found, and this is still a further object a of our invention, that submitting some l7a-rnethy1-androstenols of the normal and 19-nor-series to the above-mentioned fermenting process with Nocardia italz'oa u.sp., the androgenic properties of the starting 16-desoxy-steroid are so remarkably reduced as to make them negligible, while the 16u-hydroxy-steroids obtained show a remarkable activity in inhibiting the experimental tumors.

The products of the invention which have proved useful'in the therapy of tumors are: 4,16oc-dlhYdfOXY-17otmethyl-testosterone and its 4,16-diacyl-derivatives, 4,16- dihydroxy-l7m-methyl1 9-nor-testosterone and its 4,16-diacyl-derivatives, 16oz hydroxy 17oz methyl 19 nortestosterone and its 16-acyl-derivatives. The starting 16- desoxy-steroid for the preparation of 4,16u-dihydroxy- 17a methyl testosterone is 4 hydroxy 17o: methyltestosterone (described in the British Patent No. 848,288), while the starting 16-desoxy-steroicl for the preparation of 4,16oc dihydroxy 17a methyl 19 nor testosterone is 4-hydroxy-17a-methyl19-nor testosterone (described in the British Patent No. 888,665) and the 16- desoxy-steroid for the preparation of 16e-hydroxy-l7utmethyl-19-nor-testosterone is 17a-methyl l9-norstestosterone. Among the above-mentioned compounds the only product known in the literature is 16a-hYClI'OXY-l7w1116tl1- yl-19-nor-testosteroue, described by Seymour and E. W. Cantrall (J. Org. Chem., 26, 1961, page 3560), who report only a very weak androgenic property but without any mention of an anticancer power.

These products, have an anticancer activity and have shown especially valuable in inhibiting Ehrlichs adenocarcinoma and Ehrlichs ascitis tumor in mice. As will be further demonstrated, the products of the present invention, administered to mice, inoculated with Ehrlichs sadenocarcinoma or Ehrlichs aseitis tumor cause the regression of tumor without any deleterious eifect. They are administered preferably by a subcutaneous route as a suspension or solution in a suitable organic diluent such as polyethylene glycols, mineral oils, complex esters and ltensioactive.

The following examples serve to illustrate, but are not intended to limit, the present invention.

' EXAMPLE 1 A culture or" N. iralica aged 5 days on glucose-potato- .agar was employed to inoculate two 300 cc. Erlenmeyer flask was kept in arefrigerator for 20 hours, then the flasks each containing 60 cc. of the following medium; Casein g... 2 -Dextrin I g V 20 Corn steep g 3 Calcium carbonate, CaCO g 4 Ammonium sulfate (NI-19 80 *g r 1 Potassium hydrogen phosphate, K HPO4 g 0.1 Glucose V g 10 Tap water a 1000 6.6

H Sterilization: 120 C. for 20 minutes.

The flasks were incubated at 28 C. for 40 hours on a rotary shaker with a range of 6 cm. at 220 rpm. 6 cc. of the culture thus obtained were used to inoculate ten 300 cc. flasks, each containing 60 cc, of the following medium:

Sterilization: 120" C. for 30 minutes.

60 mg. of 9a-fluoro-hydrocortisone, dissolved in 0.5 cc.

of dimethylformamide were then added to the flasks. The flasks :were incubated at 28 C. on a rotary shaker similar to that described for the preparation of the vegetative culture.

A chromatogarphic test, carried out after 3 days of incubation, showed the disappearance of the starting prodnot and the formation of a product having an Rf equal to that of 16a-hydroxy-9a-fluoro-hydrocortisone. The contents of the flasks were then combined, and 100 g. of

'Ce'lite were added thereto and the resulting mass was warmed up to 60 C. with stirring for 10 minutes. The

" mycelium cake was then filtered and washed with 100 cc.

of water at 60 C. The filtrate Wasextracted 3 times with ethyl acetate. The ethyl acetate extracts were combined, Washed once with a saturated solution of sodium bicarbonate, then twice with distilled water. The organic extract was evaporated in vacuo to reduce the volume to 7 about 150 cc. of residual solution, which was transferred into .a smaller flask and evaporated to a volume of 30 00.; at this stage an abundant crystallization took place. The

product was filtered and washed with cold ethylacetate. 0.460 g. of 16a-hydroxy 9a-fluoro-hydrocortisone were obtained, melting at .240-245 C.; [a] =+l0O (c.=1 in methanol). i

Upon recrystallization from methanol ethyl acetate, 0.400 g. of l6a-hydroxy-9a fluoro-hydrocortisone were obtained, melting at 248-2'50 C.; (c.'== 1 in methanol). Yields 80%. i

EXAMPLE 2 The fermentation was carried out as in Example 1 while using a medium of the following composition:

Sterilization: C. for 20 minutes. 7

A chromatographic test showed the disappearance of the starting product after 4 days of incubation; A product with an Rf equal to that of 16a-hydroxy-9a fiuoro-hydrocortisone was obtained. Extraction with ethyl acetate and subsequent concentration until crystallization begins to give pure 16a-hydroxy-9a-fluoro-hydrocortisone in a yield of 75% EXAMPLE 3 3 liters of the vegetative medium as in Example 1 were sterilized at 120 C. for 60 minutes in a 5 1.laboratory fermenter. They were then inoculated with 30 cc. of culture broth in a flask as described in Example 1 and thereafter incubated for 24 hours at 28 C. with stirring with afour-paddle propellerat a rate of 400 rpm. and at an aerating rate of 3 liters per minute.

5.4 liters of the fermentation medium as described in Example 1 are sterilized in a 10 l. laboratory fermenter and inocualted with 600 cc. of vegetative medium. 6 g. of 9oc-flt1010-hYdIOCO1tlSOI16 dissolved in 35 cc. of dimethylformamide were added under sterile conditions to the medium and incubated at 28 C. with stirring with a four-paddle propeller at a rate of 450 rpm. and under an aerating rate of 5 liters per minute. 1 I r A chromatographic test showed the disappearance of the starting compound after 65 hours of fermentation. Extraction with ethyl acetate and subsequent concentration gave 16a-hydroxy-9a-fluoro-hydrocortisone in a yield of 80%. V

EXAMPLE 4 The fermentation was carried out as in Example 1 using 9m-fluoro-prednisolone as the starting material. As soon as the chromatographic test showed that 9oc-flt1010- prednisolone was completely transformed, the usual extraction was performed, whereby l6oL-l1YdlOXY-9ozfluoro-prednisolone melting at 248250 C. was obtained. On recrystallization from ethyl acetate-methanol the pure product melting at 260-262" C. was obtained. [u] =+63 (c.=l in methanol).

EXAMPLE 5 EXAMPLE 6 The fermentation was carried out as in Example 1 using progesterone (60 mg.) as the starting steroid. On recrystallization from ethyl acetate 16a-hydroxy-progesterone 7 melting at 212214 C. was obtained. [a] =+48 (c.=1 in chloroform).

EXAMILE 7 The fermentation was carried out as in Example 1 using 60 mgaof testosterone. After the extraction the product was chromatographed over a silica gel column by elution with ether containing of acetone to give 16m-hydroxytestosterone, melting at 182184 C.; (c.=1 in methanol).

EXAMPLE 8 The fermentation was carried out as in Example 1 using Reichsteins Compound S as the, starting material. Upon extraction with ethyl acetate, evaporation to dryness and crystallization from acetone-ether 4-pI'gl'l6l'l-160(,170t,21- triol-3,20-dione was obtained; melting point 235-240 C.; [m] =+93 (c.== 1 in methanol).

EXAMPLE 9 EXAMPLE 10 The fermentation was carried out as in Example 1 using 60 mg. of desoxycorticosterone acetate as the starting material. Upon extraction with ethyl acetate and crystallization from dichloroethane, 16a-hydroxy-desoxycorticosterone, melting at 202204 C., was obtained. In this case, Nocardia italz'ca, besides introducing a hydroxy group into the 16OL-PGSitiOI1, also saponified the 21-acetate-group.

EXAMPLE 11.16a-HYDROXY-17u\IETHYL19-NOR- TESTOSTERONE The fermentationwas carried out as in Example 1,

using 17a-methyl-19-nor-testosterone as the starting ma-.

terial. Upon extraction with ethyl acetate, evaporation to dryness and recrystallization from acetone-ethyl ether, 16cc hydroxy 17a methyl 19 nor testosterone was obtained, melting at 205208 C., [a] =-.-7 (c.=1 in dioxane);

g g f at 241 my; illm. 524

EXAMPLE 12.-1 6a-HYDROXYl7a-METHYL19-NOR- TESTOSTERONE The preparation was carried out in the same way as in Example 11, difiering only in the extraction technique. More exactly, the extraction of the fermentation broth was carried out with methyl-isobutyl-ketone without previously removing the mycelium, then by chromatograph- 8 ing the dry extract over Florisil, an activated magnesium silicate; 16oz hydroxy 17o. methyl 19 nor testosterone was eluted with ethyl-ether containing 10% of acetone. The 16a-acetyl-derivative, obtained in known manner with acetic anhydride in pyridine, melts at 177- 180 C.; [oc] =--17 (c.=1 indioxane)? 240 my; 47s

EXAMPLE 13.4,16a-DIHYDROXYJ7nMETHYL- TE STOSTERONE The preparation was carried out in the same Way as in Example 1, from 4-hydroxy-17oc-methyl-testosterone.

4,16 dihydroxy 17a methyl testosterone was obtained, melting at 226228 C.; [a] =|36.2 (c.=1 in clioxane);

ne 277 m 3 3 By reacting said compound with an acylating agent in known manner the corresponding 4,16-diacyl-derivatives were obtained. In this manner 4,16-diacetate, 4,16- dipropionate, 4,16-dibenzoate, and other 4,16-diacyl-derivatives were obtained.

EXAMPLE 1at.al,16a-DIHYDROXYiTaJH'ETHYLJD-NOR- TESTOSTERONE.

The preparation was carried out in the same way as in Example 1 from 4-hydroxy-l7a-methyh19-nor-testosterone. 4,16 dihydroxy 17oz methyl 19 nor testosterone was obtained, which crystallized with difiiculty. Byacetylating with acetic anhydride in pyridine, in known manner, the corresponding 4,16e-diacetate was obtained, melting at 156l58 C.; [a] =1 (c.-=1 in dioxane);

mat at 246 my; 131%,, 385

EXAMPLE l5.-PHARMACOLOGY In the following table the androgenic and anticancer activities of 4,16a-dihydroxy-17u-methyl-19-nor-testosterone, of 4,IGa-dihydroxy-17a-methyl-testosterone and of lfia-hydroxy-lh-rnethyl-19-nor-testosterone are reported in. comparison with the starting.16-desoxy-steroids. Their androgenic activity was determined according to the method of Hershoerger et al. (Proc. Soc. Exp. Biol. and Med, 83, 1953, page 175) and the anticancer activity was tested on Ehrlichs adenocarcinoma. The values of the androgenic activity are referred to testosterone propionate, conventionally taken as equal to 100, while the values of the anticancer activity are listed as percentage of inhibiting the increase of Ehrlichs adenocarcinoma.

The products were administered subcutaneously to mice as described above. The table shows that 160chydroxy-steroids, prepared according to the present invention, present a negligible androgenic activity and an anticancer activity remarkably higher than that of the starting 16-desoxy-ster0ids.

Table II Steroids Activity Androgenic Anticancer 4 hydroxy 17o: methyl 19 nortestosterone.

4,160; dihydroxy 17a methyl 19- nor-testcsterone. 4-hydroxy-lM-methyltestosterone 4,160: dihydroxy 17a methyltestosterone.

Wet-methyl-lQ-nor-testosterone. 16a liydroxy 17a methyl 19 nor testosterone.

{45.8%, 300 mg./kg./day 10 (lays. 39.2%, 225 me/kgJdayxfi days.

37%, 135 mg./kg./day 7 days.

19.4%, 300 mgJkg/rlayxg days. 20%, 143 mg./kg./day 3 days.

38.6%, 275 Ing./kg./day 10 days. 36.8%, 92.5 mg./kg./day 10days.

Very slight" 22.5 Very slight.

17.4 Very slight.

9 10 We claim: 3. 4,16a dihydroxy 17 on methyl testosterone 4,16- 1. Steroid compounds having the following formula: diacetate.

4. 4,16a-dihydroXy-17a-methyl-19-nor-testoster0ne. f 5. 4,16 dihydroxy 17oz methyl 19 nor testos- E 5 terone-4,16-diacetate. l References Cited by the Examiner R UNITED STATES PATENTS 2,666,016 1/54 Hechter et al. 195-51 10 2,756,179 7/56 Fried et al. 19551 2,936,312 5/60 Babcock et a1 260-397.4 L 2,937,192 5/60 Colton 260-3974 3,031,445 4/62 Szpilfogel et a1 2607-23955 wherern R 1s selected from the group conslstmg of hydrogen and methyl; R is selected from the group con- 15 OTHER REFERENCES sisting of hydrogen and a radical selected from the group Le d et l J A113 75, 2943.48 (1954),

consisting of an aliphatic, cycloaliphatic and aromatic acid having from 1 to 9 carbon atoms. LEWIS GOTTS Pr'mary Exammer' 2. 4,l6a-dihydroxy-17a-methyl-testosterone. M. LIEBMAN, Examiner. 

1. STEROID COMPOUNDS HAVING THE FOLLOWING FORMULA: 